Protein phosphorylation is widely accepted as one of the principal mechanisms in the control of almost all cellular processes. Recent studies have provided evidence that protein phosphorylation plays a major role in the regulation of neuronal function. A newly discovered unique class of protein kinases which exclusively phosphorylates the tyrosine residues of their substrate proteins has been shown to be abundant in nervous tissue. The determination of the role of tyrosine-specific protein kinases in the regulation of neuronal function is the major goal of this research proposal. To accomplish this goal, the tyrosine phosphorylation of the nicotinic acetylcholine receptor will be studied and used as a model system for the regulation of neurotransmitter receptors and ion channels by tyrosine kinases. The nicotinic acetylcholine receptor is a neurotransmitter-regulated ion channel and is the most well-characterized neurotransmitter receptor and ion channel in biology today. The specific aim of this project is to identify the tyrosine kinase(s) that phosphorylate(s) the nicotinic receptor in vitro and in vivo and to determine the functional consequences of tyrosine phosphorylation of the receptor. To accomplish this goal the tyrosine kinase(s) from postsynaptic membranes enriched in the nicotinic receptor will be purified and biochemically characterized. The nicotinic receptor will be phosphorylated on tyrosine and the sites of phosphorylation determined by protein sequencing techniques. Receptor phosphorylated on tyrosine residues will be purified and reconstituted into phospholipid vesicles. The functional properties of the reconstituted phosphorylated receptor will then be analyzed. In addition, the tyrosine phosphorylation of the nicotinic acetylcholine receptor in intact muscle cell will be studied and the regulation of this phosphorylation investigated. The functional effects of the tyrosine phosphorylation in muscle will also be analyzed. The proposed research project will provide a better understanding of the role of a basic regulatory mechanism in the modulation of neuronal function. Such knowledge is critical for the understanding of both normal and abnormal neuronal function.